Hindawi Publishing Corporation

International Journal of Microbiology

Volume 2016, Article ID 5127515, 7 pages
http://dx.doi.org/10.1155/2016/5127515

Research Article
Physicochemical and Antibacterial Properties of Chitosan
Extracted from Waste Shrimp Shells

José Carlos Vilar Junior,1,2 Daylin Rubio Ribeaux,2,3 Carlos Alberto Alves da Silva,2
and Galba Maria De Campos-Takaki2
1
Northeast Network for Biotechnology, Federal Rural University of Pernambuco, 52171-900 Recife, PE, Brazil
2
Nucleus of Research in Environmental Sciences and Biotechnology, Catholic University of Pernambuco,
50050-590 Recife, PE, Brazil
3
Doctorate Program in Biological Sciences, Federal University of Pernambuco, 50670-420 Recife, PE, Brazil

Correspondence should be addressed to Galba Maria De Campos-Takaki; galba takaki@yahoo.com.br

Received 4 February 2016; Accepted 3 April 2016

Academic Editor: Joseph Falkinham

Copyright © 2016 José Carlos Vilar Junior et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

This research aims to study the production of chitosan from shrimp shell (Litopenaeus vannamei) of waste origin using two chemical
methodologies involving demineralization, deproteinization, and the degree of deacetylation. The evaluation of the quality of
chitosan from waste shrimp shells includes parameters for the yield, physical chemistry characteristics by infrared spectroscopy
(FT-IR), the degree of deacetylation, and antibacterial activity. The results showed (by Method 1) extraction yields for chitin
of 33% and for chitosan of 49% and a 76% degree of deacetylation. Chitosan obtained by Method 2 was more efficient: chitin
(36%) and chitosan (63%), with a high degree of deacetylation (81.7%). The antibacterial activity was tested against Gram-negative
bacteria (Stenotrophomonas maltophilia and Enterobacter cloacae) and Gram-positive Bacillus subtilis and the Minimum Inhibitory
Concentrations (MIC) and the Minimum Bactericidal Concentration (MBC) were determined. Method 2 showed that extracted
chitosan has good antimicrobial potential against Gram-positive and Gram-negative bacteria and that the process is viable.

1. Introduction a cationic polyelectrolyte due to the protonation of amine

Chitin is the most abundant organic substance in nature
after cellulose. This biopolymer has a highly linear structure
and is insoluble in water. It dissolves quickly in concentrated
acids and in some fluoroalcohols yet its reactivity is low.
Other relevant properties of this biopolymer are its high
molecular weight and porous structure which favors high
water absorption [1, 2]. On the other hand, chitosan is
obtained from this biopolymer and this has a structural
function. These associations act as a matrix that interacts with
other constituents such as phenolic tannins in insects and
minerals in the carapaces of crustaceans [3, 4].
Deacetylation is the nonenzymatic process whereby chi-
tosan is obtained by removing R-NHCOCH3 residue and
treating it with strong alkali at high temperatures. When the
degree of deacetylation is greater than 50%, the biopolymer
becomes soluble in acidic aqueous solutions and behaves as
groups in the presence of H+ ions [5, 6]. Other ways to obtain
chitosan are by using enzymatic processes. However, they are
not used on an industrial scale owing to the high commercial
cost of enzymes (deacetylases) and their low productivity,
while nonenzymatic chemical processes are widely used
because the cost of doing so is low and the processes are
efficient [7, 8].
The development of new applications for chitosan is
strongly based on the fact that this polymer can be obtained
from renewable sources such as fisheries; it is nontoxic,
nonallergenic, biodegradable, and present in antimicrobial
activity [9].
Studies with planktonic crustaceans such as Daphnia
longispina resting eggs indicate that these crustaceans can be
exploited as a source of chitin due to their high chitin content
(23∼25%) [10]. Leptinotarsa decemlineata, also known as the
Colorado potato beetle, is a major pest of potato crops.
2 International Journal of Microbiology
The adult and larva have 20% and 7% of chitin content,
respectively. However, the chitin from adult Colorado potato
beetles had a more stable structure than that from the larvae.
Investigation has indicated that the adult potato beetle is
more appropriate as a chitin source, both because of its chitin
and chitosan content and because of its antimicrobial and
antioxidant activities [11].
Kaya et al. [12] conducted studies on potential sources
of chitin in the Orthoptera order of insects Calliptamus
barbarus and found this to be 20.5 ± 0.7% and 16.5 ± 0.7%
for Oedaleus decorus, the yield of chitosan being 74–76%,
with a deacetylation degree of 70–75%. The insects showed
potential as alternative sources of chitin and chitosan on
account of their antimicrobial and antioxidant properties for
the food/animal feed industry.
Among the most common applications are their uses
as complexing material metal ions, such as edible coatings
with antifungal and bactericidal action [13–15] and as a basic
element for making controlled drug delivery matrices [16].
Thus, the objective of this study was to investigate the
efficiency of different methodologies to obtain chitosan from
the waste of Litopenaeus vannamei shrimps since this raw
material comes from renewable resources and it is econom-
ically viable to produce high-value added compounds from
it.
2. Materials and Methods
2.1. Preparation of Raw Material. Shrimp residues of the
species named as Litopenaeus vannamei were washed in
running water and a 2.5% hypochlorite solution. Thereafter,
they were dried at room temperature and then crushed and
passed through a 16-mesh knit.
2.2. Microorganisms. The bacteria Stenotrophomonas mal-
tophilia (UCP-1600), S. maltophilia (UCP-1601), Bacillus sub-
tilis (UCP-1002), and Enterobacter cloacae (UCP-1603) were
kindly supplied from the Culture Collection UCP (Univer-
sidade Católica de Pernambuco), Recife, PE, Brazil. Culture
Collection is registered in the WFCC (World Federation
Culture for Collection). These microorganisms were used in
the tests of evaluation of Minimum Inhibitory Concentration
(MIC) and the Minimum Bactericidal Concentration (MBC).
The microorganisms were maintained at 25∘ C in nutrient agar
medium (peptone 0.5%, beef extract 0.3%, NaCl 0.5%, agar
1.5%, and distilled water, and pH is adjusted to neutral 7.4).
2.3. Chitin and Chitosan Extraction. The extraction of chitin
and chitosan was performed according to the methods
described by Zamani et al. [17] (Method 1) and Arantes
[18] (Method 2). In order to eliminate the proteins of the
residue, NaOH solutions 0.5 M (30 : 1 v/m, 90∘ C, 2 h) and
0.3 M (10 : 1 v/m, 80∘ C, 1 h, under agitation), respectively, were
used. Then, the alkali-insoluble fraction (IFA) was separated
by centrifugation at 4000⋅g for 15 minutes and/or by vac-
uum filtration. Subsequently, to demineralize the precipitate
obtained, 10% acetic acid (100 : 1 v/m, 60∘ C, 6 h) and 0.55 M
hydrochloric acid (10 : 1 v/m, room temperature, 1,5 hours)
were used. To obtain purified chitosan, treatments with 1%
sulfuric acid (121∘ C/20 min) and 50% NaOH (100∘ C, 10 h)
were performed.
2.4. Spectroscopy to Infrared Ray (IR). Two milligrams of
chitin and chitosan samples was dried overnight at 60∘ C
under reduced pressure; then, this was homogenized with
100 mg of KBr. Discs with the prepared KBr were dried
for 24 h at 110∘ C under reduced pressure. The chitin and
chitosan samples from shrimp shell (Litopenaeus vannamei)
waste were analyzed at 4000–625 cm−1 using an infrared
ray Fourier Transform Spectrometer (FT-IR, BRUKER Mod.
IFS). A KBr disc was used as reference. To determine the
maximum absorption intensity of bands, the baseline was
used.
2.5. Determination of the Deacetylation Degree (DD%). The
degree of acetylation and deacetylation of chitosan was deter-
mined using an infrared ray spectroscopy, IR 22, applying the
band 𝐴 1655 /𝐴 3450 which was calculated as per [19]
𝐴 1655 100
𝐴𝐷 (%) = ( )× . (1)
𝐴 3450 1,33
2.6. Evaluation of Minimum Inhibitory Concentration (MIC).
To evaluate the Minimum Inhibitory Concentration (MIC),
the serial dilution technique was used with the tested
microorganisms, in accordance with Qi et al. [20]. An initial
chitosan solution was prepared at 0.5% in 1% acetic acid
and pH = 5.0. Then, serial dilutions were performed of
1 : 1 to 1 : 512 and decreasing concentrations ranging from
0.00005% to 0.25%. For each microorganism, a standard
bacterial suspension was prepared in sterile saline solution
and compared to the 0.5 McFarland scale tube. 10 𝜇L bacterial
suspension was transferred to each one in the series of tubes
and incubated at 37∘ C for 24 hours.
2.7. Evaluation of Minimum Bactericidal Concentration
(MBC). For the evaluation of Minimum Bactericidal Con-
centration (MBC), a qualitative technique was used according
to the method of Qi et al. [20]. The series of chitosan solutions,
which determined the MIC, were used to evaluate MBC.
From the reading of the MIC, the tubes that showed no visible
turbidity had 10 𝜇L plated on blood agar, pH 7.0, and were
incubated for 24 h at 37∘ C, and observations were made on
whether or not the colonies of microorganisms grew.
3. Results and Discussion
According to elementary studies and analyses of different
crustaceans (shrimp, lobster, and squid), there was great
variability of this composition when chitin amounts were
varied for squid of approximately 1.8%, pink shrimp 22%
(under study), and lobster 36% [21]. Hence, there is a need
to develop efficient demineralization and deproteinization
processes to remove mineral content (20–30%) and protein
content of approximately 40% in order to obtain chitin that
is free of inorganic and protein content. This study showed
International Journal of Microbiology
Table 1: Chitin and chitosan copolymers obtained by the proposed methodologies.
Method Shell waste (g)
Chitin (%)
Method 1 10 33
Zamani et al. [17]
Method 2 10 36
Arantes [18]
Table 2: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of chitosan by Zamani et al. [17] and
Arantes [18] on Gram-positive and Gram-negative bacteria.
Method 1
Microorganisms
MIC/𝜇g⋅mL−1
S. maltophilia (UCP-1600) 156
S. maltophilia (UCP-1601) 78
B. subtilis (UCP-1002) 625
E. cloacae (UCP-1603) 78
that different concentrations of NaOH and demineralization
with hydrochloric acid and acetic acid influenced the yield
of the extraction process used to obtain chitin and chitosan.
Similarly, it was proved that the methods used also had an
effect on the degree of deacetylation (Table 1). To confirm that
the biopolymer was chitosan, the product obtained with the
commercial chitosan Sigma (Sigma Aldrich Corp., St. Louis,
MO, USA) was characterized and compared by infrared
spectrometry.
The residual mass from shrimp exoskeleton after dem-
ineralization and deproteinization processes showed well
preserved chitin structure as described by Stamford et al.
[22]. This was higher than the values obtained by Tenuta
Filho and Zucas [23], with 14% of chitin pink shrimp waste
(Penaeus brasiliensis) and by Beaney et al. [24] with 10% yield
of biopolymer from Nephrops norvegicus.
Kaya et al. [25] found that the chitin content of bat guano
species Rhinolophus hipposideros collected from Karacamal
Cave was 28%. It was noted the chitosan productivity cor-
responding to 79% from isolate chitin is superior to our
results from L. vannamei using two different methodologies.
However, the chitosan content from L. vannamei using two
methods showed better results.
A new chitin source was described by Kaya et al. [26]
using Gammarus argaeus Crustacea. The results showed the
isolation of alpha chitin and were confirmed by infrared spec-
troscopy, thermogravimetric analysis (TGA), X-ray diffrac-
tion (XRD), and scanning electron microscopy (SEM) tech-
niques.
More recently, the production of a new morphology of
chitin from the wings of Periplaneta americana has been
studied by Kaya and Baran [27]. They showed the surface
of the chitin has oval nanopores (230–510 nm) without
nanofibers. The chitin surface had a pore in the center and six
or seven other pores distributed around these, corresponding
to structures similar to cell walls. Alternatively, studies with
chitin content of the structure of the exoskeleton of seven
species from grasshopper of the four genera were carried out
3
Copolymers
Degree of deacetylation (%)
Chitosan (%)
49 76.0
63 81.7
Method 2
MBC/𝜇g⋅mL−1 MIC/𝜇g⋅mL−1 MBC/𝜇g⋅mL−1
312 312 312
156 78 156
1250 625 625
156 156 156
[28]. The contents of chitin varied between 5.3% and 8.9%
and had a low molecular weight (between 5.2 and 6.8 kDa). A
large amount of waste is formed from invasive and harmful
species that have been killed by the use of insecticides, and
the authors suggest that these be collected and evaluated as
an alternative chitin source.
Some parameters in the deacetylation reaction are cited
as fundamental factors on the end characteristics of chitosan
[29]. Tsaih and Chen studied the influence of temperature
and processing time on polymer chitosan characteristics and
found that both have a significant effect on the deacetylation
degree and molecular weight [30].
The results obtained also showed a higher yield than that
found for the chitin extracted from shrimp Penaeus brasilien-
sis [31], which was 5.3% and 2.5% of chitosan. Santos et al.
[32] showed a lower percentage with 5.9 and 5.06% of chitin
and chitosan, respectively. Thus, the maximum chitosan
obtained from chitin deacetylation (57.5%) was similar to the
reported value for the extraction from the polymer using
the shrimp Macrobrachium rosenbergii (∼65%). However, the
results obtained by Battisti and Campana-Filho showed that
80% of chitin was transformed into chitosan [33].
The spectrophotometric analysis of commercial chitosan
(Figure 1(a)) and the chitosan obtained by the methods used
(Figures 1(b) and 1(c)) enabled the bands to be characterized
as follows: Peak 1 (∼1650 cm−1 ) corresponded to acetylated
residues (NHCOCH3 ) of chitosan; Peak 2 (∼1590 cm−1 )
identified the NH2 groups present in the deacetylate residues;
and Peak 3 (3440 cm−1 ) corresponded to the vibration of the
OH molecule [34]. Analysis by FT-IR estimated the amount
of free amine groups present in the molecule of chitosan
obtained from the two methodologies, namely, 76% and
81.7%, respectively (Table 2). However, the higher deacetyla-
tion degree of chitosan is generally controlled by processing
the native polymer with alkali and increasing time and
temperature [30]. These values are consistent with commer-
cial chitosan, obtained from crustaceans, since this reaches
between 75 and 90% deacetylation in industrial processing.
4 International Journal of Microbiology

1,1
1,00
1,0

0,95 0,9
12
0,8 12

T (%)
T (%)

0,90 3
3
0,7
0,85
0,6
0,80 0,5

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
(cm−1 ) (cm−1 )
(a) (b)
1,1

1,0

0,9
3 1
0,8 2
T (%)

0,7

0,6

0,5

4000 3500 3000 2500 2000 1500 1000 500

(cm−1 )
(c)

Figure 1: Infrared absorption spectra: (a) commercial chitosan (Sigma Aldrich Corp., St. Louis, MO, USA); (b) chitosan obtained by Method
1 [17]; (c) chitosan obtained by Method 2 [18]. The peaks in chitosan were indicated as 1 = (∼1650 cm−1 ) acetylated residues (NHCOCH3 );
2 = (∼1590 cm−1 ) NH2 groups present in the deacetylate residues; and 3 = (3440 cm−1 ) the vibration of the OH molecule.

In a study proposing a simple and efficient method
of deacetylation of chitosan using acetate of 1-butyl-3-
methylimidazole, as the reaction catalyst, DD% = 86 was
obtained, a value similar to that found in our study in the best
condition for producing the biopolymer (DD = 81.7%) [35].
Santos et al. [32] determined the degree of deacetylation
of chitosan obtained from the shrimp “Saburica” (Macro-
brachium jelskii), which was approximately DD 76%, using
linear potentiometric titration. The results using Fourier
transform infrared spectroscopy obtained in this study are
in agreement with the data in the literature, which may vary
from 50 to 92.3% [36, 37].
Hennig [31] analyzed obtaining chitosan from Penaeus
brasiliensis and obtained a degree of deacetylation (DD%) of
87%. This value was similar to that reported in the literature
for Weska et al. [38] and to those obtained in the best
condition.
Furthermore, it was shown that the chitosan produced
has characteristics comparable to commercial chitosan, the
degree of deacetylation ranging between 70 and 95% [36, 39].
Recently, Kaya et al. [40] undertook studies on chitin
obtained from Insecta (Melolontha melolontha) and Crus-
tacea (Oniscus asellus) and compared their physical and
chemical properties. The results showed chitin content for dry
weights of M. melolontha and O. asellus corresponding to 13-
14% and 6-7%, respectively. The results observed that chitin
nanofibers of O. asellus adhered to each other; nanofibers of
M. melolontha were nonadherent and were considered the
more attractive chitin source.
Studies were carried out with Fomitopsis pinicola, a
medicinal fungus in Asia, and found 30.11% of chitin and yield
of 71.75% of chitosan from the dry weight. The chitin showed
acetylation of 72.5% and deacetylation of chitosan was 73.1%,
and the maximum chitin temperature of degradation was
341∘ C [41]. Results clearly revealed a significant deacetylation
degree of chitosan from waste shrimp shell Litopenaeus
vannamei using two methodologies in comparison with
deacetylation degree of chitosan determined to F. pinicola
[41].
Fourier transform infrared spectroscopy (FT-IR), ele-
mental analysis (EA), thermogravimetric analysis (TGA), X-
ray diffractometry (XRD), and scanning electron microscopy
(SEM) were used to investigate chitin structure isolated from
both sexes of four grasshopper species, and it was observed
that the amount of chitin was greater in males than females
[42].
The physicochemical properties of chitin are investigated
and related to taxonomy. The results showed those chitins
International Journal of Microbiology
properties are affected in different parts of the body (head,
thorax, abdomen, legs, and wings) of the honey bee related
to the extraction method. Physical and chemical properties
form a parameter involved with taxonomy, and the chitin
extracts from different parts of the body are different [43].
The influence of chitosan extracted by the methods pro-
posed on inhibiting the growth of Stenothrophomonas mal-
tophilia (UCP-1600/UCP-1601), Enterobacter cloacae (UCP-
1603), and Bacillus subtilis (UCP-1602) is shown in Table 2.
The actual mechanism of inhibition is not fully understood.
However, the more acceptable hypothesis is related to a
change in the permeability of the cell due to interactions
between the biopolymer chitosan, when it is positively
charged (pH below 6.5), and the cell membrane of microor-
ganisms when negatively charged [44].
In the present study, the results demonstrated that MIC
and MBC were more significant in Gram-negative bacteria
when compared with Gram-positive ones but were effective
in both cases. These results are in agreement with reports in
the literature that have documented antimicrobial activity of
chitosan against a large number of microorganisms, the MIC
ranging between 0.1% and 1% [45, 46]. Thus, the efficiency of
the chemical-physical characteristics is also related, as well as
species or strains of bacteria in the same study [47, 48].
Wang [49] demonstrated that, for bactericidal action of
chitosan on E. coli, solutions with concentrations between
0.5 and 1% at 48 hours had to be used, and to obtain the
same effect at 24 h, higher concentrations of 1% chitosan were
prepared. In addition, Tsai and Su [50] demonstrated that
solutions of chitin and chitosan of high molecular weight
and a high degree of deacetylation had a lethal effect on E.
coli and Shigella dysenteriae when concentrations between
50 and 500 ppm were used. According to Chung et al. [47],
the hydrophilicity of the cell wall and the negatively charged
cell surface was greater in Gram-negative bacteria in relation
to Gram-positive bacteria. In addition, the distribution of
negative charges on their cell surfaces was very different when
compared with Gram-positive bacteria, thus supporting the
results found in this study.
In a study conducted to evaluate the bactericidal activ-
ity of glucose-chitosan complex of E. coli, Pseudomonas,
Staphylococcus aureus, and Bacillus cereus, it was determined
that the Minimum Inhibitory Concentration of chitosan was
around 0.05%, these results being the same as those found
in this study [51]. Moreover, on using the chitosan extracted
from Rhizopus arrhizus and Cunninghamella elegans to eval-
uate the MIC and MBC on Listeria monocytogenes, Staphylo-
coccus aureus, Pseudomonas aeruginosa, Salmonella enterica,
Escherichia coli, and Yersinia enterocolitica, it was observed
that the MIC values ranged from 200 𝜇g⋅mL−1 for E. coli to
500 𝜇g⋅mL−1 of L. monocytogenes. However, for the MBC, the
results were between 400 𝜇g and 1000 𝜇g/mL−1 , respectively
[52]. The values determined in this study corroborate the
results shown in Table 2. Thus, in this research study, the
effect of the chitosan obtained by the proposed methods
was proved to be effective as an antimicrobial agent on the
microorganisms tested. Hence, the efficiency of applying this
biopolymer in the therapeutic area was confirmed.
5
4. Conclusion
The previous results recommend Method 2 for the chemical
reaction as it offers a clean, cheap, and convenient method
for extracting chitosan from chitin extracted from shrimp
wastes. Within the results in this work, the conclusion was
reached that shrimp wastes are an excellent source for chitin.
The yields and acetylation degree of chitosan decreased
the concentration of NaOH solution, the temperature, and
the length of treatment. The chitosan obtained showed the
highest degree of deacetylation. Different chitosans were
tested and markedly inhibited the growth of most bacteria
tested; however, the inhibitory effects differed depending on
the types of chitosan and the bacteria tested, there being
greater antimicrobial activity against Gram-positive bacteria
than against Gram-negative bacteria.
Competing Interests
The authors declare no conflict of interests.
Acknowledgments
This research study was supported financially by CNPq
(National Council for Scientific and Technological Devel-
opment, Brası́lia, DF, Brazil), FACEPE (Foundation for the
Support of Science and Technology of the State of Pernam-
buco, Recife, PE, Brazil), and CAPES (Coordination for the
Improvement of Higher Level Education, Brası́lia, DF, Brazil).
The authors are also grateful to the Nucleus of Research
in Environmental Sciences and Biotechnology (NPCIAMB),
the Catholic University of Pernambuco (Recife, PE, Brazil),
for the use of its laboratories, and PROCAD-CAPES, from
AESGA.
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